Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Reexamination Certificate
1999-03-11
2002-02-05
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
C436S073000, C436S079000, C436S086000, C436S087000, C436S093000, C436S095000, C436S166000, C436S172000, C548S110000, C534S015000
Reexamination Certificate
active
06344360
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to fluorescent compounds useful as indicator molecules for detecting the presence or concentration of an analyte in a medium, such as a liquid, and to methods for achieving such detection. More particularly, the invention relates to fluorescent lanthanide metal chelate complexes containing substituted ligands and their use as indicator molecules for detecting the presence or concentration of an analyte such as glucose or other cis-diol compound in a medium, including a liquid medium such as a biological fluid.
2. Description of the Related Art
Certain rare-earth metal chelates emit visible light upon irradiation with UV light and different forms of visible light (e.g., violet or blue light), an emission which is characterized by the chelated cation. Some lanthanide ions, such as those of europium (Eu
3+
), samarium (Sm
3+
), terbium (Tb
3+
), and to a lesser extent dysprosium (Dy
3+
) and neodymium (Nd
3+
), exhibit typical fluorescence characterized by the ion, especially when chelated to suitable excitation energy mediating organic ligands. The fluorescent properties of these compounds—long Stokes' shift, narrow band-type emission lines, and unusually long fluorescence lifetimes—have made them attractive candidates for fluorescent immunoassays and time-resolved fluorometric techniques.
The major emission lines of these fluorescent lanthanide chelates are formed from a transition called hypersensitive transition and are around 613-615 nm with Eu
3+
, 545 (and 490) nm with Tb
3+
, 590 and 643 nm with Sm
3+
, and 573 with Dy
3+
. See Hemmila,
Application of Fluorescence in Immunoassays
, 140-42 (1991). See also
Spectroscopy in Inorganic Chemistry
, vol. 2, at 255-85 (Academic Press 1971). Radiation is typically absorbed by the chelates at a wavelength characteristic of the organic ligand and emitted as a line spectrum characteristic of the metal ion because of an intramolecular energy transfer from the ligand to the central metal ion. The organic ligand absorbs energy and is raised or excited from its singlet ground state, S
0
, to any one of the vibrational multiplets of the first singlet excited state, S
1
, where it rapidly loses its excess vibrational energy. At this point, there are two possibilities: relaxation by an S
1
→S
0
transition (ligand fluorescence) or intersystem crossing to one of the triplet states, T
1
. See E. P. Diamandis et al.,
Analytical Chemistry
62:(22):1149A (1990); see also
Spectroscopy in Inorganic Chemistry
, vol. 2, at 255-85 (Academic Press 1971).
Fluorescent europium chelates are known to exhibit large Stokes shifts (~290 nm) with no overlap between the excitation and emission spectra and very narrow (10-nm bandwidth) emission spectra at 615 nm. In addition, the long fluorescence lifetimes (measurable in microseconds instead of the nanosecond lifetimes measurable for conventional fluorophores) of the chelates help filter out noise and other interference having a low fluorescent lifetime. The long fluorescent lifetimes thus permit use of the chelates for microsecond time-resolved fluorescence measurements, which further reduce the observed background signals. Additional advantages of using europium chelates include that europium chelates are not quenched by oxygen.
Line emissions of two europium (Eu) chelates, Eu-dibenzoylmethide and Eu-benzoylacetonate, have made the chelates attractive candidates for use in lasers. See H. Samuelson, et al. (
J. Chem. Physics
39(1): 110-12 (1963)) Samuelson, et al. studied the fluorescence and absorption of the above two europium chelates as solids and in solution. Samuelson, et al. compared the fluorescent lifetimes of the europium chelates under various conditions with the lifetimes of europium fluorescence in other compounds. Based on this comparison, Samuelson, et al. suggested that the variation in lifetimes between the two groups of europium compounds is a result of the ligand-Eu interaction in the europium chelates. Specifically, Samuelson et al. determined that various emission lines from Eu-dibenzoylmethide showed fluorescent lifetimes of 480 +/−50 &mgr;s, which were significantly greater than the fluorescent lifetimes in other europium compounds.
Crosby, et al.,
J. Chem. Physics
34:743 (1961) had previously studied the role of intramolecular energy transfer in sensitizing ion emission from rare-earth metal chelates, including europium dibenzoylmethide and europium benzoylacetonate chelates. Whan, et al.,
J. Mol. Spectroscopy
8: 315-27 (1962) reported that the emission from chelates of a group of lanthanide metal ions (Eu
3+
, Tb
3+
, Dy
3+
and Sm
3+
) was dominated by bright spectral lines characteristic of the individual rare-earth metal ions. Whan, et al. found that both the benzoylacetonates and dibenzoylmethides of Eu
3+
and Tb
3+
are especially bright emitters and that the bright line emissions and low yields of phosphorescence from these chelates indicated that intramolecular energy transfer from the ligands to the Eu
3+
and Tb
3+
ions of these chelates occurs efficiently. Whan, et al., at 324.
N. Filipescu, et al.,
J. Physical Chem
. 68(11):3324 (1964) reported that the fluorescence spectra of europium and terbium &bgr;-diketone chelates are modified when substituents are changed in the organic ligand portion of the chelates. Filipescu, et al. discussed the relative intensity, spectral distribution, shifting, and splitting of the fluorescence lines of the europium and terbium chelates in relation to the nature of substituents, their position, molecular configuration, and the overall intramolecular energy transfer. Filipescu, et al. found that the overall fluorescence intensity characteristic of the ion depended on two factors: 1) the amount of energy available at the organic triplet, and 2) the efficiency of energy transfer to the ion.
Filipescu, et al. also found that the above two factors varied for different substituents. For instance, the substitution of europium dibenzoylmethide chelates with electron-donor methoxy groups in the meta position on the chelate was found to enhance the fluorescent emission of the europium ion, whereas paramethoxy substitution was found to decrease the europium fluorescence. Additionally, the effect was more pronounced for the di- than for the monomethoxy-substituted dibenzoylmethides. In contrast, an opposite effect was observed for nitro-substituted dibenzoylmethides of europium. The electron-withdrawing nitro groups attached to the para or meta positions were found to decrease the total ionic emission of europium. Additionally, the effect was more pronounced for di- than for monosubstituted dibenzoylmethides.
Filipescu, et al. further found that the strong ionic fluorescence emitted by europium para-phenyldibenzoylmethide indicated that increasing the size of the aromatic system enhanced the amount of energy transferred to the europium ion. This fact was confirmed by the emission results obtained for napthyl-substituted diketones which were found to have substantially higher ionic emissions than the dibenzoylmethide chelates. Filipescu, et al., at 3328-29.
E. Diamandis, et al.,
Analytical Chemistry
62(22): 1149A (1990), described how europium chelates can be used as labels in fluorescence immunoassays and DNA hybridization assays. With respect to fluorescent immunoasays, the authors described that europium chelates can be used as immunological labels in various assay configurations, including either competitive or noncompetitive assays.
U.S. Pat. No. 4,374,120 (Soini, et al.) describes a method for detecting a substance using a fluorescent lanthanide chelate complex as a marker. U.S. Pat. No. 4,374,120 also describes the use of &bgr;-diketones as enhancing ligands for promoting the strong fluorescence properties of certain lanthanide chelates, especially chelates of europium and te
Colvin Arthur E.
Daniloff George Y.
Kalivretenos Aristole G.
Nikolaitchik Alexandre V.
Parker David
Rothwell Figg Ernst & Manbeck
Sensors for Medicine and Science, Inc.
Soderquist Arlen
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